Glycoconjugate vaccines are widely recognized for their ability to prevent many life-threatening bacterial infections. Glycoconjugate vaccines are generally considered efficacious and safe and have been used in humans for over 30 years. Conventional glycovaccine production often involves the chemical modification of immunogenic carrier proteins with polysaccharide antigens of pathogenic bacteria. However, more recently, biotechnological processes for producing glycoconjugate vaccines have emerged that are expected to reduce production costs and to further increase the homogeneity and possibly the potency and safety of glycoconjugate vaccine preparations.
In eukaryotic cells, N-linked glycosylation is a key posttranslational protein modification mechanism involving several enzymes. In prokaryotic cells N-linked glycosylation is catalyzed by certain bacterial N-oligosaccharyltransferases (N-OSTs). The protein glycosylation gene cluster of Campylobacter jejuni (C. jejuni) includes the pglB gene, which encodes a membrane-bound N-OST (PglBCj). PglBCj can be expressed in standard bacterial hosts, such as Escherichia coli (E. coli), and can glycosylate co-expressed periplasmic proteins that carry at least one surface-exposed D/E-Y-N-X-S/T (Y, X≠P) glycosylation motif. PglBCj can transfer bacterial polysaccharide antigens to C. jejuni proteins as well as to immunogenic carrier proteins of other organisms containing engineered glycosylation sites. PglBCj can transfer C. jejuni oligosaccharides and, to a certain degree, O-antigen lipopolysaccharide structures of Gram-negative bacteria and capsular antigen polysaccharides of Gram-positive bacteria.
The present disclosure provides recombinant N-OSTs with modified substrate specificities and methods of using the recombinant N-OSTs for glycoconjugate vaccine production. Such recombinant N-OSTs can advantageously be used in N-glycosylation of proteins.